Development of ERE-Transgenic Zebrafish for Studying Health Effects of Environmental Oestrogens

View/Open

Date

Author

Date issued

Type

Language

Publisher

Reason for embargo

We are trying to publish paper, but we need to do more works for it. I think it will take around 2 years including lab works, submission abd reviews from the journal.

Abstract

It is now well established that there are a wide variety of known EDCs (Endocrine Disrupting Chemicals) in the aquatic environment, which include natural (e.g. oestrogen) and synthetic hormones (e.g. ethinyloestradiol), weak environmental oestrogens (e.g. bisphenol A and nonylphenol) and pesticides, fungicides and herbicides, that are able to alter the physiology of exposed wildlife and humans and have become a focus of increasing concern for human and environmental health over the past two last decades. However, the functional implications and potential significance of these findings has yet to be established. In this thesis, a transgenic fish system approach (using a new plasmid) was employed to investigate the effect pathways of EDCs in fish, using transgenic UAS-GFP zebrafish (Danio rerio) as a model species.
One of the most critical steps in this approach is the development of an effective construct for the development of sensitive transgenic zebrafish. The required ERE was cloned and three copies were incorporated into a construct. Three vector systems (pKS+, pCS2+ and pBR322-Tol2) were selected for making new plasmids, and investigated to improve the efficiency for sensitivity and tissue specificity. Three constructs were made and tested progressively in the development process. The plasmids contain three oestrogen response elements (3ERE), a TATA box and GAL4ff. The three constructs, pKS-ERE-Gal4ff, pCS2-ERE-Galf4ff and pBR-Tol2-ERE, were examined by injecting into zebrafish in a transient assay system. The pBR-Tol2-ERE-Gal4ff construct was selected for generating transgenic zebrafish because it showed tissue specific manner (i.e. reduce mosaicism) and Tol2 transposon has the capacity to improving greatly the chances for generating TG fish such as high germline transmission frequency.
The construct pBR-Tol2-ERE-Gal4ff required to examine the functional capability of using transient expression assay before generating transgenic fish. The Gal4-UAS/GFP method was adopted and produced a two step amplification of the oestrogenic signal. A novel transient expression assay system was developed using a synthetic oestrogen responsive element, the Tol2 mediated Gal4-UAS systems and the GFP reporter gene, which are responsive to environmental oestrogens using green fluorescent microscopy in zebrafish (Danio rerio) and medaka (Oryzias latipes). The construct worked well and vector system in transient expression assays proved a rapid, sensitive, tissue specific system for the detection of oestrogenic EDCs in both the zebrafish and medaka, indicating the likely wide suitability for application to other fish species. These data indicate that this system would be a particularly powerful technique for use in species with long generation times and/or where there are other difficulties for generating transgenic lines in those species.
After the transient expression assay, the construct pBR-Tol2-ERE-Gal4ff with transposase mRNA was co-injected into early one cell stage and bred for 3 months. The embryos were collected from the founders (a male and a female) and exposed to EE2 to identify the transgenic zebrafish. An oestrogen responsive transgenic zebrafish (ERE-TG fish) was established under an oestrogen inducible promoter derived from multiple tandem oestrogen responsive elements. Gal4ff-UAS system was adopted to generate ERE-TG fish to enhance the sensitivity, which has not been used for generating biosensor zebrafish yet. Oestrogen exposures were shown to induce specific GFP expression in the heart, muscle, otic vesicle, brain, neuromasts, eye/ear ganglions and fin, in addition to that in the liver and gonad, which has not been shown previously in other oestrogen responsive transgenic zebrafish, illustrating both the enhanced sensitivity of our detection system and the potential for these tissues as target sites for wider health effects for exposure to environmental oestrogens. Furthermore, oestrogen chemicals are converted to an oestrogenic signal in different tissues preference. Oestrogen receptors (ER α, β1 and β2) morpholinos (MOs) and exposures with oestrogen receptor antagonist were carried out to assess whether the GFP expression was mediated through the ER, regardless of the subtypes. GFP expression was inhibited through injection of the mixture of ERs MOs compared with uninjected transgenic fish larvae. This result indicated that GFP expression was mediated through the ERs. The lowest detectable concentration of EE2, E2, BPA and NP was 1 ngEE2/L, 5 ngE2/L, 100 µgBPA/L and 1 µgNP/L in ERE-TG fish embryos/larvae. These findings provide that ERE-TG fish embryos/larvae systems are useful both for studying physiological mechanisms and for detecting biological target sites of environmental oestrogens.
As a preliminary experiment, immature (40 day old) and mature (3 month old) ERE-TG fish were exposed to low concentrations of EE2 for 7 days to prove its potential for developing tissue-specific models of EDCs at different life stages. The lowest detectable EE2 effect concentration was 5 ngEE2/L in the liver and gonad and 10 ngEE2/L in the muscle in both immature and mature ERE-TG fish. These data demonstrate that muscle is as susceptible as liver and gonad to EE2 and ERE-TG fish (both immature and mature fish) would appear to be a useful system for both detecting target sites for oestrogen chemicals in these life stages fish.
In summary, the work undertaken in this thesis has developed transiently and stably transfected zebrafish as a tool for screening oestrogen chemicals to assess for the potential health impacts of oestrogens in the environment, provided novel insights into many tissue target tissues by oestrogen exposure, and has established different patterns of tissue responses for different environmental oestrogens tested suggesting differing functional implications.